Protein expression, purification, and characterization
Introduction:
The production of recombinant proteins is an important aspect in biological research. Introducing a DNA of interest into an organism’s existing DNA can create Recombinant DNA. The goal is to express a certain gene that is not already present in an organism. The first step in making a recombinant protein involves cloning the gene of relevance. The gene is amplified by PCR and this product is then cloned into a specific expression system (i.e. vectors such as plasmids). The plasmid is then inserted into a host cell, which is most commonly a bacterium. Bacteria cultures are grown to express the specific protein and the cells are harvested for further study. To make sure the bacteria has indeed taken up the plasmid, selective markers are added to help identify the transformed bacteria. For example, antibiotic resistance is a type of selective marker that can be used to harvest only the cells that have been transformed. The gene that has ampicillin resistance is cloned along with the plasmid into the host cell. This way, only the bacteria that have the ampicillin resistance gene will survive. Once the cells are harvested, the next step is to purify the protein that is expressed in the bacterial cell. Protein purification involves lysing the bacterial cell to release the soluble proteins. The protein must be separated from extraneous material such as cell debris. The lysate is clarified by centrifugation and the protein is purified by adding a tag that attracts the protein (chromatography). Using a second binding/elution process such as ion exchange chromatography can further purify the protein. Samples of the purified protein are collected and freezed for further experiments. The last step is protein characterization, which involves using gel electrophoresis to analyse the protein samples. The proteins will be separated because of mass differences and the distance of travel through the gel. The samples can also be characterized by UV/VIS spectroscopy. The concentration is calculated by measuring the absorbance value at 280nm and manipulating Beer's Law. These concentration values are compared to the results from gel electrophoresis and conclusions can be drawn upon the whole experiment. In this particular experiment, the purple protein pGEM-gbr22 is collected from the Great Barrier Reef and is expressed in the bacteria.
Methods:
25 ul of bacteria was added to 2 tubes each. 2 ul of plasmid (pGEB-gbr22) is added to one of the tubes, which will be the DNA tube, and the other tube will serve as the control. The tubes are placed in an ice bath for 30 min and heat shocked for 45 seconds in 42 degree Celsius water bath. 200 ul of SOC media is added to the tubes and are placed in the incubator for 30 min at 37 degree Celsius at 250rpm. The bacteria mixture is then spread onto both the plates and is stored overnight in the incubator at 37 degrees Celsius. A single bacterial colony is added to LB broth and is left in the incubator for about 6 to 7 hours. Then, 25ml of the starter culture is added to an Erlenmeyer flask and the appropriate amount of ampicillin was added to make the final concentration 100ug/ml. This is left in the incubator to grow for 16 to 24 hours. Retrieve a sample (Sample 1). The culture is then centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius. The pellet is retrieved and the liquid is dispensed. The pellet is suspended in 1x PBS solution (2.5ml added to the pellet). Then, lysozyme is added to make the final concentration 1mg/ml. The tube is then placed in a freezer at -20 degrees Celsius overnight. The solution is then placed in the incubator for 20 min at room temperature to ensure complete lysis. Then, 2 ul of benzonase is added to the solution and is incubated for another 15 minutes at room temperature. The solution is then centrifuged for 20 min at 14,000 rpm at 4 degrees Celsius. Retrieve a sample after the centrifugation (Sample 2). The liquid is collected and the pellet is thrown out. The liquid (lysate) is syringe filtered. Two buffers are made; a wash buffer with 1x PBS and 20mM imidazole (final concentration of 10ml) & an elution buffer with 1x PBS and 250mM imidazole (10 ml final concentration). The protein is then purified using batch and column chromatography. 0.5 ml of Ni-NTA resin/buffer is added to the column. The protein is added to the tube and is left in there to incubate for 15 minutes. Then, a sample is collected of the flow through (Sample 3). Next, 5 ml of wash buffer is allowed to flow through the column and a 4th sample is collected. Then, 5ml of elution buffer is allowed to flow through and a 5th sample is retrieved. Lastly, the remaining amount of elution buffer is added to the column and a final sample is collected (6th Sample). Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) is used to measure the absorbance of the elution 1 sample (Sample 5) at both 280nm and 574nm. This is done in both normal and UV/VIS mode. The samples are then loaded onto s SDS-PAGE gel for gel electrophoresis. The MW standard used was PageRuler by Fermentas. The loading buffer was made with 6x gel loading buffer and a purple stain was added to the samples before it was loaded in the gel. The gel electrophoresis lasted for 25 minutes at 200V. Then, 30 ml of imperial protein stain was added to the gel and was left in an orbital shaker for 1 to 1.5 hours. The gel was destained a few times and left overnight to remove any background staining. Lastly, the gel was dried at 75 degrees Celsius for 1.5 hours.
Results:
Figure 1: LB agar with amp: Experimental with DNA, purple colonies of E.coli were grown.Transformed with gbr22 plasmid DNA.
Figure 2: LB agar plate with Amp. There was no DNA in this plate so no bacteria growth was present.
Figure 3: Fun plate was coughed on. There was no ampicillin in this plate to begin with. There was no bacterial growth.
Figure 4: This is our purple culture. The culture was left over night to make a larger culture.
Figure 5: This is our harvested cells in a form of a purple pellet. The weight of the pellet A was 0.44g and the weight of pellet B was 0.40 grams.
Figure 6: Elution 1 and 2 are shown. Elution 1 contains the protein gbr 22 as indicated by the purple color. The 2nd elution does not contain protein
Calculations for absorbance values: Beer’s Law: UV/VIS: A= εlc @ 280nm .054= (38850L/mol x cm) (1cm) (c) C= .355 mg/ml A= εlc @ 574nm (maximum wavelength) 10(.067) = (118300L/mol x cm) (1cm) (c) C=.145mg/ml Check document below to see the figures obtained from spectrophotometry.
Figure 7: Gel containing the protein fragments prior to drying. A total of 7 lanes present. The first lane is the PageRuler Molecular Weight Standard and the following 6 lanes contain the 6 samples respectively.
Figure 8: The "dry" gel. Shown are the 7 lanes-1 MW standard lane and the other six lanes contain the samples.
Figure 9: PageRuler by Fermentas MW standard.-protein ladder
The atomic weight of the protein using the standards as the reference is 25kDa.
Discussion: The concentration obtained at 280nm and the maximum wavelength should have theoretically been closer together because it is assumed that the solution is purified. This disparity might have been due to the fact that the entire wash buffer was used in the column chromatography instead of the entire elution buffer. Also, the lyase was not incubated for 15 minutes and this could have inhibited the nickel beads from binding to the histine tag of the protein. As far as the gel electrophoresis results, the estimated atomic weight of the protein was on par with the actual atomic weight of the protein. The atomic weight turned out to be 25kDa in both cases. However, the elution 1 sample (5) was not pure because there were 3 bands of equal intensity which meant that there were other proteins present besides the protein of interest. Again, this could have been due to incorrect method of purification used. Conclusion: pGEM-gbr22 protein was expressed in bacteria and was isolated to do future experiments regarding its enzymatic activity. The steps used for this experiment include protein expression, purification, and characterization. The next step in this experiment involves performing enzyme assays to learn about the proteins function and try to study inhibitory behavior of the protein when it is bound to the ligands discovered from virtual drug screening. References: 1. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008,5 (2), 135-46
Protein expression, purification, and characterization
Introduction:
The production of recombinant proteins is an important aspect in biological research. Introducing a DNA of interest into an organism’s existing DNA can create Recombinant DNA. The goal is to express a certain gene that is not already present in an organism. The first step in making a recombinant protein involves cloning the gene of relevance. The gene is amplified by PCR and this product is then cloned into a specific expression system (i.e. vectors such as plasmids). The plasmid is then inserted into a host cell, which is most commonly a bacterium. Bacteria cultures are grown to express the specific protein and the cells are harvested for further study. To make sure the bacteria has indeed taken up the plasmid, selective markers are added to help identify the transformed bacteria. For example, antibiotic resistance is a type of selective marker that can be used to harvest only the cells that have been transformed. The gene that has ampicillin resistance is cloned along with the plasmid into the host cell. This way, only the bacteria that have the ampicillin resistance gene will survive. Once the cells are harvested, the next step is to purify the protein that is expressed in the bacterial cell. Protein purification involves lysing the bacterial cell to release the soluble proteins. The protein must be separated from extraneous material such as cell debris. The lysate is clarified by centrifugation and the protein is purified by adding a tag that attracts the protein (chromatography). Using a second binding/elution process such as ion exchange chromatography can further purify the protein. Samples of the purified protein are collected and freezed for further experiments. The last step is protein characterization, which involves using gel electrophoresis to analyse the protein samples. The proteins will be separated because of mass differences and the distance of travel through the gel. The samples can also be characterized by UV/VIS spectroscopy. The concentration is calculated by measuring the absorbance value at 280nm and manipulating Beer's Law. These concentration values are compared to the results from gel electrophoresis and conclusions can be drawn upon the whole experiment. In this particular experiment, the purple protein pGEM-gbr22 is collected from the Great Barrier Reef and is expressed in the bacteria.
Methods:
25 ul of bacteria was added to 2 tubes each. 2 ul of plasmid (pGEB-gbr22) is added to one of the tubes, which will be the DNA tube, and the other tube will serve as the control. The tubes are placed in an ice bath for 30 min and heat shocked for 45 seconds in 42 degree Celsius water bath. 200 ul of SOC media is added to the tubes and are placed in the incubator for 30 min at 37 degree Celsius at 250rpm. The bacteria mixture is then spread onto both the plates and is stored overnight in the incubator at 37 degrees Celsius. A single bacterial colony is added to LB broth and is left in the incubator for about 6 to 7 hours. Then, 25ml of the starter culture is added to an Erlenmeyer flask and the appropriate amount of ampicillin was added to make the final concentration 100ug/ml. This is left in the incubator to grow for 16 to 24 hours. Retrieve a sample (Sample 1). The culture is then centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius. The pellet is retrieved and the liquid is dispensed. The pellet is suspended in 1x PBS solution (2.5ml added to the pellet). Then, lysozyme is added to make the final concentration 1mg/ml. The tube is then placed in a freezer at -20 degrees Celsius overnight. The solution is then placed in the incubator for 20 min at room temperature to ensure complete lysis. Then, 2 ul of benzonase is added to the solution and is incubated for another 15 minutes at room temperature. The solution is then centrifuged for 20 min at 14,000 rpm at 4 degrees Celsius. Retrieve a sample after the centrifugation (Sample 2). The liquid is collected and the pellet is thrown out. The liquid (lysate) is syringe filtered. Two buffers are made; a wash buffer with 1x PBS and 20mM imidazole (final concentration of 10ml) & an elution buffer with 1x PBS and 250mM imidazole (10 ml final concentration). The protein is then purified using batch and column chromatography. 0.5 ml of Ni-NTA resin/buffer is added to the column. The protein is added to the tube and is left in there to incubate for 15 minutes. Then, a sample is collected of the flow through (Sample 3). Next, 5 ml of wash buffer is allowed to flow through the column and a 4th sample is collected. Then, 5ml of elution buffer is allowed to flow through and a 5th sample is retrieved. Lastly, the remaining amount of elution buffer is added to the column and a final sample is collected (6th Sample). Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) is used to measure the absorbance of the elution 1 sample (Sample 5) at both 280nm and 574nm. This is done in both normal and UV/VIS mode. The samples are then loaded onto s SDS-PAGE gel for gel electrophoresis. The MW standard used was PageRuler by Fermentas. The loading buffer was made with 6x gel loading buffer and a purple stain was added to the samples before it was loaded in the gel. The gel electrophoresis lasted for 25 minutes at 200V. Then, 30 ml of imperial protein stain was added to the gel and was left in an orbital shaker for 1 to 1.5 hours. The gel was destained a few times and left overnight to remove any background staining. Lastly, the gel was dried at 75 degrees Celsius for 1.5 hours.
Results:
Calculations for absorbance values:
Beer’s Law:
UV/VIS:
A= εlc @ 280nm
.054= (38850L/mol x cm) (1cm) (c)
C= .355 mg/ml
A= εlc @ 574nm (maximum wavelength)
10(.067) = (118300L/mol x cm) (1cm) (c)
C=.145mg/ml
Check document below to see the figures obtained from spectrophotometry.
Sample 1 - cell lysate
Sample 2 - after centrifugation
Sample 3 - flow through
Sample 4 - wash
Sample 5 - Elution 1
Sample 6 - Elution2
The atomic weight of the protein using the standards as the reference is 25kDa.
Discussion:
The concentration obtained at 280nm and the maximum wavelength should have theoretically been closer together because it is assumed that the solution is purified. This disparity might have been due to the fact that the entire wash buffer was used in the column chromatography instead of the entire elution buffer. Also, the lyase was not incubated for 15 minutes and this could have inhibited the nickel beads from binding to the histine tag of the protein. As far as the gel electrophoresis results, the estimated atomic weight of the protein was on par with the actual atomic weight of the protein. The atomic weight turned out to be 25kDa in both cases. However, the elution 1 sample (5) was not pure because there were 3 bands of equal intensity which meant that there were other proteins present besides the protein of interest. Again, this could have been due to incorrect method of purification used.
Conclusion:
pGEM-gbr22 protein was expressed in bacteria and was isolated to do future experiments regarding its enzymatic activity. The steps used for this experiment include protein expression, purification, and characterization. The next step in this experiment involves performing enzyme assays to learn about the proteins function and try to study inhibitory behavior of the protein when it is bound to the ligands discovered from virtual drug screening.
References:
1. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46
2. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/ (accessed April 18, 2011).2.